From Fatty to Functional: Developing Defatting Strategies to Enhance Steatotic Liver Transplant Outcomes

Liver disease affects approximately 4.5 million individuals in the United States (US) and accounts for an estimated 55,000 deaths annually. Liver transplantation remains the only definitive treatment for end-stage liver disease. Since the early 2010s, liver transplants have been on the rise, nearing 10,000 per year as of 2021. However, there is a shortage of viable organs due to insufficient donation numbers and the simultaneous increase in the rate of donor liver discard. Liver steatosis is the primary reason for discard, accounting for approximately 40-60% of all unused livers. As steatotic liver disease (SLD) affects 25% of the global population, increasing parallel to obesity rates, the liver discard rate is projected to increase to 66% by 2030 if no interventions are implemented. One approach for liver rehabilitation is defatting steatotic organs ex vivo prior to their transplantation. Normothermic machine perfusion (NMP) is an emerging technology that allows for this potential intervention. However, the bulk of the work to rehabilitate steatotic organs has relied on murine models and a defatting cocktail made of reagents with limited safety data, potential cytotoxicity, and risks of carcinogenesis, making them unsuitable for clinical translation. Moreover, attempts to integrate this defatting cocktail into a NMP system have been successful in murine models, but these results have yet to translate to human steatotic livers.

The goals of this dissertation were therefore: 1) to develop a reproducible human in vitro model of hepatic steatosis that closely mimics the pathophysiological features of steatotic liver disease, providing a platform for studying lipid metabolism and testing therapeutic interventions, 2) to develop a tool capable of measuring lipid sizes from maximum intensity projections of lipid images, enabling more precise characterization of the lipid accumulation in lipid-laden cells from immunofluorescent images, 3) to design and optimize a pharmacological defatting cocktail capable of effectively reducing the lipid content within a human steatosis model while preserving hepatic viability and functionality, and 4) to explore antioxidant strategies for reducing oxidative stress in steatosis, potentially enhancing the safety and efficacy of future defatting protocols.

In this work, the construction of a three-dimensional human steatosis model considered not only the important cell types but also the pathological, environmental cues that contribute to the disease progression in vivo. After optimization, steatosis induction resulted in a mix of macro- and micro-steatosis, consistent with the human pathology. This steatotic model was then utilized as a drug testing platform whereby reagents were systematically tested for their defatting efficacy and preservation of viability. Maximal defatting was achieved through simultaneously targeting lipolysis, lipophagy, and β-oxidation. Multi-omics analyses revealed a mechanistic shift in metabolism, with an upregulation of PPARa-mediated b-oxidation and an efflux of saturated fatty acids and cholesterol. However, it was found that during this process, defatted organoids were enhancing their responses to reactive oxygen species (ROS). In order to combat this, the application of antioxidants was tested as a means to reduce general ROS in steatotic liver organoids and explore their potential as an adjunct therapy in managing oxidative stress and improving outcomes in steatotic liver transplants. Taken together, this research advances our understanding of hepatic steatosis and offers approaches to improve the viability of steatotic livers for transplantation